Our research is focused on elucidating the mechanisms that underlie ATP-dependent protein remodeling carried out by molecular chaperone machines and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target regulatory proteins and misfolded proteins for degradation. During cell stress, chaperones play an essential role in preventing folding intermediates from becoming irreversibly damaged and forming protein aggregates. They promote recovery from stress by disaggregating and reactivating proteins, a process once thought to be impossible. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation, misfolding and premature degradation are major contributors to a large number of human diseases, including cancer, Alzheimer's, Parkinson's, type II diabetes, cystic fibrosis, and prion diseases. The goal of our research is to understand how chaperones function and to provide the foundation for discovering preventions and treatments for diseases involving protein misfolding. One aim is to understand the mechanism of action of Hsp90. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of more than 200 client proteins, including receptors, protein kinases and transcription factors. Hsp90 is also important for the growth and survival of cancer cells and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of this important family of chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec, and from yeast, Hsp82. Using a novel phenotype associated with Hsp90Ec overexpression in E. coli, we selected for Hsp90Ec mutants with defective chaperone function. We identified a functional region of Hsp90Ec that contains surface-exposed residues from the middle and C-terminal domains. To understand the role of this region, we purified the Hsp90Ec mutant proteins and characterized them in vitro. In contrast to wild type Hsp90Ec that reactivates heat-denatured luciferase in collaboration with the DnaK chaperone system, the Hsp90Ec mutants were defective for chaperone function. The mutant proteins hydrolyzed ATP, however, unlike wild type Hsp90Ec, the ATPase activities of the mutant proteins were poorly stimulated by client proteins suggesting that they bound clients weakly. Results from protein binding assays demonstrated that the Hsp90Ec mutant proteins were defective for client protein binding. Thus, our results define a functional region in E. coli Hsp90 that is important for substrate binding. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90. A second aim is to elucidate the mechanism of protein disaggregation by Clp/Hsp100 molecular chaperones, including ClpB of prokaryotes and its yeast homolog, Hsp104. Understanding how energy-dependent protein disaggregating machines function is universally important and clinically relevant since protein aggregation is linked to medical conditions like Alzheimer's disease, Parkinson's disease, amyloidosis and prion diseases. ClpB/Hsp104 dissolves insoluble aggregated proteins in combination with a second molecular chaperone system, DnaK in E. coli and Hsp70 in yeast. In the absence of the DnaK/Hsp70 system, ClpB and Hsp104 have the intrinsic ability to disaggregate soluble aggregates in vitro. Conformational changes in ClpB/Hsp104 driven by ATP binding and hydrolysis promote substrate binding, unfolding and translocation. Conserved pore tyrosines in both nucleotide-binding domain-1 (NBD-1) and 2 (NBD-2) that reside in flexible loops extending into the central pore of the ClpB/Hsp104 hexamer, bind substrates. When the NBD-1 pore loop tyrosine is substituted with alanine (Y251A), ClpB can collaborate with the DnaK system in disaggregation, although activity is reduced. The N-domain has also been implicated in substrate binding and like the NBD-1 pore loop tyrosine, is not essential for disaggregation activity. To further probe the function and interplay of the ClpB N-domain and the NBD-1 pore loop, we made a double mutant with an N-domain deletion and Y251A substitution. This ClpB double mutant is inactive in substrate disaggregation with the DnaK system, although each single mutant alone can function with DnaK. Our data suggest that this loss in activity is primarily due to a decrease in substrate engagement by ClpB prior to substrate unfolding and translocation, and indicate an overlapping function for the N-domain and NBD-1 pore tyrosine. Furthermore, the functional overlap seen in the presence of the DnaK system is not observed in the absence of DnaK. For innate ClpB unfolding activity, the NBD-1 pore tyrosine is required, and the presence of the N-domain is insufficient to overcome the defect of the ClpB Y251A mutant. We have further investigated the role of the ClpB N-domain in aggregate binding in collaboration with Michal Zolkiewski's laboratory (Kansas State University). It was suggested that the N-terminal domain's mobility that is maintained by the unstructured linker connecting the N-domain and NBD-1 might control the efficiency of aggregate reactivation. Several variants of ClpB with modified sequence of the N-terminal linker were constructed. The mutant proteins showed lower rates of disaggregation and reactivation than wt ClpB. These results suggest that the linker does not merely connect the N-terminal domain, but supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. A third aim is to investigate the mechanism of action of Clp chaperones in proteolysis. Clp proteases of prokaryotes have analogous structures, functions and mechanisms of action to the eukaryotic proteasome. They are composed of an ATP-dependent protein unfolding component and a protease component. Some are regulated by adaptor proteins and antiadaptor proteins. We are currently studying ClpX, which associates with a proteolytic component, ClpP, forming the ClpXP ATP-dependent protease. In an ongoing collaboration with Susan Gottesman's laboratory (NCI), we are studying how ClpXP is regulated by adaptor and anti-adaptor proteins. RssB is a ClpXP adaptor protein that specifically targets RpoS, the stationary phase sigma factor of E. coli, for degradation during exponential growth. In response to stress, RpoS degradation ceases. Three small anti-adaptor proteins, IraP, IraM, and IraD, are each made under a different stress condition and interact with RssB to block RpoS degradation. We have been studying the reaction in vitro with purified proteins. By characterizing wild type and mutant RssB proteins we are gaining insight into the mechanism of action of the RssB adaptor and how the interaction of RssB with antiadaptor proteins inhibits the action of RssB.